THE DETERMINATION OF GLYCOGEN IN LIVER AND

MUSCLE BY USE OF ANTHRONE REAGENT*

BY NICHOLAS V. CARROLL, ROBERT W. LONGLEY, AND

JOSEPH H. ROE
(From the Department of Biochemistry, School of Medicine,
George Washington University, Washington, D. C.)

(Received for publication, October 28, 1955)

In currently used methods for the determination of glycogen, tissue is

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extracted either by boiling with 30 per cent potassium hydroxide solution
(KOH) or by homogenization with trichloroacetic acid solution (TCA).
The glycogen is precipitated from the extract by alcohol and determined
either by copper reduction after separation by centrifugation, acid hydroly-
sis, and neutralization (l-3) or by direct treatment of the precipitated
glycogen with anthrone reagent (4-6). An alternative procedure is to
destroy alkali-labile carbohydrate by boiling with KOH and then deter-
mine the glycogen in the alkali-treated mixture with anthrone reagent
(5, 7).
Previously, we have reported that a method based upon the use of an-
throne reagent gave results of a high degree of specificity and precision (8).
This method is a modification of procedures for the determination of dex-
tran (9) and biood sugar (10). In this paper we are reporting in detail our
anthrone-adapted method, with critical studies of procedures based upon
extraction of tissues with 30 per cent KOH and with 5 per cent TCA.

Method
Reagents-
1. Anthrone reagent. A solution containing 0.05 per cent anthrone, 1
per cent thiourea, and 72 per cent by volume HzS04 is used. For each
liter of reagent, place in a suitable flask 280 ml. of distilled water and add
cautiously 720 ml. of concentrated H&04, sp. gr. 1.84, of highest purity.
Place in a flask 500 mg. of purified anthrone, 10 gm. of highest purity
thiourea, and 1 liter of the 72 per cent H,SOb. Warm the mixture to
80-90”, occasionally shaking the flask to mix the contents. Do not over-
heat the mixture. Cool and store in a refrigerator. This reagent will
keep for at least 2 weeks in a refrigerator.
2. 5 per cent trichloroacetic acid.
3:95 per cent ethanol.

* Supported in part by a grant from the Division of Research Grants and Fellow-
ships, National Institutes of Health, United States Public Health Service.

583
584 GLYCOGEN IN LIVER AND MUSCLE

4. Glucose standard. (a) Stock solution. Dissolve 100 mg. of dry, high-
est purity glucose in 100 ml. of saturated benzoic acid solution. (b) Work-
ing standard. Place 5 ml. of the stock solution in a 100 ml. volumetric
flask and make up to volume with saturated benzoic acid solution. 2 ml.
of this solution, containing 0.1 mg. of glucose, are used as a standard.
Procedure-Place the t,issue sample in an efficient blendor under an ap-
propriate volume of TCA and homogenize for 3 minutes. Pour the ho-
mogenate into a suitable centrifuge tube or bottle. Centrifuge and decant
the supernatant fluid upon an acid-washed filter paper placed in a funnel
draining into a graduated cylinder. Transfer the residue quantitatively
to the blendor with an appropriate volume of TCA and homogenize again

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for 1 minute. Centrifuge the mixture and pour the supernatant fluid
through the same filter. Two more extractions may be made in the same
manner if it is desired to extract better than 97 per cent of the glycogen
present. Make up to the desired volume with 5 per cent TCA and mix
thoroughly. The final volume should be a quantity that will contain 10
to 200 y of glycogen per ml.
1 ml. of the trichloroacetic acid filtrate is pipetted into a 15 ml. Pyrex
centrifuge tube. To obtain the most reliable results, duplicate samplesof
each unknown are analyzed. To each tube are added 5 volumes of 95 per
cent ethanol with careful blowing to effect thorough mixing. This should
be checked by noting the absence of an interface. The tubes are capped
with clean rubber stoppers and allowed to stand overnight at room tempera-
ture. (Alternatively, placing the tubes in a water bath at 3740” for 3
hours may be carried out.) After precipitation is complete, the tubes are
centrifuged at 3000 r.p.m. for 15 minutes. The clear liquid is gently de-
canted from the packed glycogen and the tubes are allowed to drain in an
inverted position for 10 minutes.
The glycogen is dissolved by addition of 2 ml. of distilled water, the water
being added in a manner that will wash down the sidesof the tube. If the
glycogen does not dissolve instantly, agitate the tube until solution is com-
plete. A reagent blank is prepared by pipetting 2 ml. of water into a clean
centrifuge tube. A standard is prepared by pipetting 2 ml. of standard
glucosesolution, containing 0.1 mg. of glucose, into a similar tube.
At this point 10 ml. of anthrone reagent are delivered into each tube
with vigorous, but consistent, blowing. The stream of anthrone reagent
is directed into the center of the tube and should be sufficient to insure good
mixing. As each tube receives anthrone reagent, it is tightly capped with
an air condenser and placed in a cold tap water bath. The air condenser
is prepared by cutting off the small end of a size 0 rubber stopper and in-
serting a 4 inch length of glass tubing, 3 to 4 mm. in diameter. This
serves to prevent water from entering the tube from the water bath.
 N. V. CARROLL, R. W. LONGLEY, AND J. ,H. ROE 585

After all tubes have reached the temperature of the cold water, they are
immersed in a boiling water bath to a depth a little above the level of the
liquid in the tubes for 15 minutes and then removed to a cold water bath
and cooled to room temperature. The tubes and stoppers are wiped dry
and the contents of each tube are transferred to a calorimeter tube and
read at 620 mp after adjusting the calorimeter with the reagent blank.
Care is taken to avoid introduction of lint or contaminating carbohydrate
into the anthrone reaction.
Calculation-The calculation of glycogen is as follows:
volume of extract
D-u x 0.1 x x 100 x 0.9
DX gm. of tissue

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= mg. of glycogen per 100 gm. of tissue
where DU = optical density of the unknown, DS = optica density of the
standard, 0.1 = mg. of glucose in 2 ml. of standard solution, 0.9 = factor
for converting glucose value to glycogen value.

DISCUSSION

Methods of Extraction-In applying anthrone reagent to the determina-

tion of glycogen the main problem was the method of extraction. Good,
Kramer, and Somogyi (2) concluded that the original Pfhiger procedure,
which involves boiling the tissue in KOH solution, alcohol precipitation,
acid hydrolysis of the precipitate, and copper reduction of the neutralized
hydrolysate, is the only adequate method for the determination of glyco-
gen. These authors introduced improvements into the Pfltiger procedure
which speeded up the method considerably.
Lower glycogen values have been found with acid extracts of tissuesthan
with extracts obtained with boiling alkaline solution (2, 6, 11, 12). Such
findings have led to the suggestion that glycogen exists in tissues in an
“easily extractable” or “free” form and a “difficult,ly extractable” or
“fixed” form (6, 13).
We made a comparative study of the acid and alkali extraction methods
applied to liver and muscle. With liver the procedure was to divide the
organ, homogenize one half with 5 per cent TCA, and treat the other half
with boiling 30 per cent KOH for 15 minutes. In experiments on muscle
one gastrocnemius muscle was homogenized with 5 per cent TCA and the
other was boiled with 30 per cent KOH. Glycogen was precipitated by
adding 1.2 volumes of 95 per cent ethanol to the KOH solution and 5 vol-
umes of et.hanol to the TCA extract. The 1.2 volumes of ethanol were
added because this concentration is employed in currently used methods
(2, 3, 5) and higher concentrations have been found to precipitate non-gly-
cogen carbohydrate (2, 3). For the precipitation from TCA solution 5
586 GLYCOGEN IN LIVER AND MUSCLE

volumes of 95 per cent ethanol were found to be optimal. The glycogen

was determined by the anthrone method described.
The results with liver, shown in Table I, are related to the level of liver
glycogen. At glycogen levels of 3 to 7 per cent the values by TCA extrac-
tion were 94 to 102 per cent of those obtained by boiling the liver with 30
per cent KOH. With fasted rats the values from homogenization with 5
per cent TCA ranged from 7 to 48 per cent of those obtained by boiling
with 30 per cent KOH.

TABLE I

Comparison of TCA and KOH Methods of Extraction of Liver

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from Fed and Fasted Rats
-
Nutritional state
I Mg. glycogen per 100 gm. liver
I gg x 100
TCA KOII Di%.XWX

Fed ........................... 2944 2980 36 99

‘I ........................... 4360 4440 80 98
“ 4330 4560 230 95
...........................
I‘ ........................... 4000 4260 260 94
“ ........................... 7162 6975 187 102
“ ........................... 3067 3233 166 95
“ ........................... 3950 4025 75 98
“ ........................... 4980 5044 64 98
Fasted ........................ 101 210 109 48
“ ........................ 42 118 76 35
‘I ........................ 15 82 67 17
‘I ........................ 103 652 549 16
“ ........................ 20 147 127 13
‘I ......................... 138 987 849 15
“ ........................ 8 107 99 8
‘I ........................ 21 307 286 7

Results of comparative studies upon muscle are shown in Table II. The
values found by TCA extraction ranged from 92 to 96 per cent of those
obtained by KOH extraction.
Bloom, Lewis, Schumpert, and Shen (6) made a study of the amounts
of glycogen obtained from liver and muscle by boiling with 30 per cent
KOH and by homogenizing with 10 per cent TCA. These authors found
that the amounts of TCA-extractable glycogen in the livers of fed rats,
rats fasted for 12 hours, and rats fasted for 24 hours were 84.9, 56.4, and
7.9 per cent, respectively, of the values obtained in each case by KOH
extraction. The per cent of TCA-extractable glycogen in liver observed
by these authors varied directly with the glycogen level. Our findings
are in good agreement with the data of Bloom et al. However, we do not,
 N. V. CARROLL, It. W. LONGLEY, AND J. H. ROE 587

agree with their assumption that the value obtained by KOH extraction
represents the “total” and therefore the true glycogen content of the liver,
since, as will be shown later, the KOH extract contains non-glycogen car-
bohydrate.
Bloom et al. (6) found that the TCA-extractable glycogen of muscle in
normal fed rats was 55 per cent of that obtained by KOH extraction, a
value that is considerably lower than the TCA recoveries we obtained
(92 to 96 per cent).
Kemp and Kits van Heijningen (12) developed a method for the determi-
nation of glycogen in which the TCA homogenate of tissue is warmed at
100” for 15 minutes. These authors found that extracts prepared with

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boiling TCA gave values for rat muscle that ranged from 89 to 111 per
cent of those obtained by the Pfliiger alkali extrachion procedure. As

TABLE II
Comparison of TCA and KOH Methods of Extraction of Muscle in Fed Rats

Mg. glycogen per 100 gm. muscle

Experiment ‘gH x 100
No.
TCA KOH Difference

1 433 453 20 96
2 493 538 45 92
3 470 507 37 93
4 651 693 42 94
5 678 717 39 94
6 567 609 42 93

shown in Table II, our recoveries of glycogen from rat muscle with cold
TCA, compared with those with KOH extraction, are as high as those ob-
tained by Kemp and Kits van Heijningen with boiling TCA, a result prob-
ably due to the high speed and repeated homogenizations we used.
Kits van Heijningen and Kemp (13) made a comparative study of the
“free” and ‘%xed” glycogen content of rat muscle. They considered the
values obtained by extracting with cold TCA as ‘<free” glycogen and those
found by extracting the cold TCA residue wit,h boiling TCA as ((fixed”
glycogen. They observed a mean ratio of “free” to “fixed” glycogen of
1.74 f 0.26. The ‘(free” glycogen thus averaged 63 per cent of the total
glycogen.
Dialysis Experiments-To obtain evidence that might explain the dis-
crepancy between the data obtained by the two methods of tissue extrac-
tion, experiments were designed to show whether anthrone-sensitive ma-
terials, other than glycogen, are present in the precipitates obtained by
treatment of the two types of extracts with alcohol.
588 GLYCOGEN IN LIVER AND MUSCLE

Livers were removed from anesthetized animals and either boiled with 30
per cent KOH or homogenized with 5 per cent TCA. To the KOH and
TCA extracts were added 1.2 and 5 volumes of 95 per cent ethanol, respec-
tively. After centrifuging, decanting, draining, and washing with 95 per
cent ethanol, the precipitates were taken up in distilled water, placed in
Visking cellophane tubing, and dialyzed. Anthrone-sensitive material
could not be demonstrated in the 24 hour dialysate from the glycogen pre-
pared by TCA extraction, but, within an hour from the starting of the
dialysis with the glycogen isolated from the boiling KOH extract, material
responding to ant#hrone reagent was found in the dialysate. The cello-

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TABLE III
Data Showing Dialyzable Anthrone-Sensitive Material in Glycogen Precipitated
from KOH Digests by 1.2 Volumes of 95 Per Cent Ethanol

I Mg. glycogen per 100 gm. liver I

Exp%:?=t Before dialysis After dialysis Difference

Per cent dialyzable

(2) (1) - (2)

2060 2040 20 1
4625 4480 145 3
3535 3400 135 4
2160 2040 120 6
1486 1358 128 9
77 57 20 26
81 58 23 28
100 60 40 40

phane used was shown to be impermeable to clinical dextran! with an av-

erage molecular weight of 70,000.
Further experiments were performed to determine t.he amount of dialyz-
able mat,erial in glycogen isolated from boiling KOH extracts. The pre-
cipitate obtained in the usual KOH procedure, after washing with 95 per
cent ethanol, was taken up in 10 ml. of water. 5 ml. of this solution were
transferred to a section of Visking cellophane tubing for dialysis and bhe
other 5 ml. were retained for a control. Dialysis was carried out against
running water for 24 hours, after which the contents of the cellophane tub-
ing were decanted into a volumetric flask, the tubing was thoroughly
washed, and the solut,ion was made up to volume. A carbohydrate deter-
mination by the anthrone method was made on the dialyzed portion and
upon the control which was handled in the same manner except that di-
alysis was omitted. The difference between the two values was considered
t’he amount of dialyzablc material. The results are shown in Table III.
These data show that the apparent glycogen precipitated by alcohol from
 N. V. CARROLL, 1~. \V. LONGLEY, AND J. 1%. ROE 589

KOH digests of liver contained water-soluble, alcohol-precipitable, an-

throne-sensitive, dialyzable material which obviously was not glycogen.
E$ect of Prolonged Boiling with KOH-The effect of prolonged boiling
of the 30 per cent KOH digests of liver was next studied. 1 ml. aliquots
of KOH extract of liver from fasted rats were pipetted into centrifuge
tubes, and the tubes were capped with condensers and placed in a boiling
water bath. At various intervals tubes were removed from the water bath
and 1.2 volumes of 95 per cent ethanol were added. 1 ml. aliquots of a
solution of purified glycogen were treated similarly. The glycogen in the
precipitates was determined by the anthrone method. The results are
shown in Fig. 1. During the 8 hour boiling period the purified glycogen

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O--e FASTED LIVER DIGEST
.55 - 0-b GLYCOGEN + KOH
o--o GLYCOGEN + KOH t BLOOD

. 25 -

.15r z 0 0

----------o------a ---------- -0
-05 L
1 I I I I I I I
I 2 3 4 5 6 7 8
TIME IN HOURS
FIG. 1. Effect of heating with 30 per cent KOH on rat liver glycogen

remained at the same concentration, but the apparent glycogen content of

the KOH digest diminished. It is well known that the content of pure
glycogen is not diminished by boiling in KOH solution. Therefore, the
decrease in the KOH digest, shown in Fig. 1, was due to decomposition of
a non-glycogen complex into fragments not precipitable by ethanol. The
decrease in apparent glycogen in this experiment was marked, because
liver from fasted rats was used in which a relatively high proportion of
non-glycogen carbohydrate is present.
Nature of Insoluble Residue in KOH Extracts-As is well known to
workers with the Pfltiger procedure, after boiling liver with 30 per cent
KOH solution, cooling, and allowing the mixture to stand for a time, an
insoluble material separates from the solution. Analysts are cautioned
not to remove t,his residue as it contains glycogen (5). This material was
examined as follows. The apparent glycogen content of the solution ob-
590 GLYCOGEN IN LIVEIL .1ND MUSCLE

tamed by boiling liver in 30 per cent KOH, after cooling but before the
insoluble residue had appeared, was determined. After the residue had
separated and the mixture was centrifuged, determinations of the glycogen
content of the supernatant fluid and the insoluble residue were made.
The results are shown in Table IV. The amount of glycogen in the super-
natant fluid plus that in the residue accounted completely for the quantity
of glycogen found in the original extract. These experiments suggested
that the insoluble residue that forms in KOH extracts of tissue is not gly-
cogen, since it is insoluble in KOH solution. However, it may be argued
that the insoluble residue is glycogen separat,ing from a supersaturated

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TABLE IV
Analysis of KOH Digests of Liver and Muscle

I Mg. per cent of apparentglycogen

Material After centrifugation
Original extract I-
Supernatant
fluid Residue

Liver. 20.4 13.9 6.8

‘I 27.4 13.4 11.1
“ 15.8
19.0 5.1
“ 39.7 16.6 20.9
“ 123.9 87.0 25.8
“ 19.2 14.3 3.9
Muscle. 567.5 552.2 17.4
‘I 717.0 705.0 16.0
“ 424.2 28.5
452.7
‘I 507.0 499.0 8.0

KOH solution. That this was not the case was shown by further study of
the nature of the residue.
It was found that this residue is not soluble in water which proved that
it was not glycogen. After washing this substance several times with
water, it was found to react characteristically with anthrone reagent, and,
after hydrolysis, to reduce alkaline copper solution. These observations
showed that the insoluble residue that forms in KOH digests of liver is not.
glycogen and is a material that responds to both anthrone and copper re-
duction techniques.
Completeness of Extraction with TCA-It is difficult to remove all of the
glycogen from tissue by grinding with TCA because of the requirement for
completely disrupting the cells and thoroughly dispersing the proteins that
are precipitated by TCA.
To study the recoveries by TCA extraction, rat livers were homogenized
 N. V. CARROLL, R. W. LONGLEY, AND J. H. ROE 591

in a Servall omnimixer at 14,000 r.p.m. The first homogenization was

for 3 minutes, after which the mixture was centrifuged and decanted. The
residue was returned to the blendor for further homogenization for 1 or 2
minutes. This procedure was repeated until five homogenizations had
been carried out. The supernatant liquid obtained aft.er each homogeniza-
tion was analyzed for glycogen. The residue after the fifth TCA extrac-
tion was taken up in 30 per cent KOH and treated by the Pfliiger proce-

TABLE V
Recovery of Glycogen from Liver by Repeated Homogenizations with 6 Per Cent
Trichloroacetic Acid with Servalb Omnimixer at 14,000 R.p.m.

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Per cent recovery
Extraction No. ‘ICime homogenized I-
5iingle extraction Cumulative
-
min. ?IIg.

1 3 3367.0 87.5 87.5

2 1 374.8 9.7 97.2
3 1 63.2 1.7 98.9
4 1 24.2 0.7 99.6
5 1 15.1 0.4 100.0
Residue by KOH 142.3
I 3 718.3 85.4 85.4
2 1 87.0 10.3 95.7
3 1 12.6 1.5 97.2
4 1 11.0 1.3 98.5
5 2 12.6 1.5 100.0
Residue by KOH 164.5
3 64.9 70.1 70.1
1 14.7 15.8 85.9
3 I 5.0 5.4 91.3
4 1 2.6 2.8 94.1
5 2 5.4 5.9 100.0
Residue by KOH
I 112.4
I
dure, the apparent glycogen being determined by the anthrone method.
The results are shown in Table V. The amounts of glycogen in the frac-
tions analyzed after the fifth homogenization, per 100 gm. of liver, were
15.1, 12.6, and 5.4 mg. for livers containing 3367,718, and 64.9 mg. per 100
gm., respectively. Since these amounts were small, it was assumed for
purposes of comparison that the glycogen obtained by the five homogeniza-
tions was all that was present. Based upon this assumption, the per cent
in each extraction is shown in the fourth column of Table V and the cumu-
lative recoveries after each extraction in the last. The cumulative re-
coveries show that 10 to 15 per cent of the total glycogen was present in
592 GLYCOGEN IN LIVER AND MUSCLE

the second extraction. A second blending of the tissue was necessary to

obtain 97,96, and 86 per cent of the glycogen with liver contents that were
average, low, and at the fasting level, respect,ively, and two more homogeni-
zations of 1 minute each were necessary to recover essentially all of t,hc
glycogen.
The liver glycogen values in Table V for the residues boiled with KOH
after five extractions with TCA are not considered true glycogen values.
The evidence from Tables II, III, and IV support this assumption.
Concerning “l+ee” and “Rxed” Glycogen-Our studies led us to the con-
clusion that the distinction between “free” and “fixed” glycogen, proposed
in certain reports in the literature, is an incorrect assumption. It is our

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opinion that the glycogen value obtained by extraction with TCA, when
the extraction procedure is adequate, is the true glycogen content of the
tissues. In our data of Table V there is no reason to assume that the gly-
cogen obtained in each successive extraction existed in the tissues in a form
differing from that in the preceding extraction, although it might be said
to be more “difficultly extractable.” Further evidence against this as-
sumption is our demonstration of the presence of non-glycogen carbohy-
drate in KOH extracts of tissues, which largely accounted for the difference
between the values obtained by TCA and KOH extractions.
Specificity of Proposed Method-The proposed method, which involves
extraction by TCA and isolation of the glycogen by alcohol precipitation,
is on a sound basis. Excellent recoveries mere obtained in analyses of
blood to which purified glycogen was added. Methods (12) in which the
TCA extract is boiled are subject to the objection that losses may occur
from hydrolysis of the glycogen.
Isolation of the glycogen by alcohol precipitation is a preferable step.
Boiling with alkali may remove interfering sugars, but usually this pro-
cedure leaves a solution that is not ideal for direct color production. Seif-
ter et al. (5) observed interference with the anthrone reaction when applied
to KOH solutions of liver from which glycogen had been removed by alco-
hol precipitation. The interference, amounting to 1.5 per cent for livers
containing 1 per cent of glycogen, was not considered serious at normal or
high glycogen levels. However, this observed error is in addition to that
produced by other interfering substances which are precipitated from KOH
digests by alcohol.
The time required for the technical manipulations involved in the pro-
posed method, in which anthrone reagent is used, is much less than that
required for copper reduction procedures.

SUMMARY

1. A method has been developed for the determination of glycogen in

liver and muscle.
 N. V. CARROLL, R. W. LONGLEY, AND J. I-1. ROE 593

2. Glycogen is precipitated from trichloroacetic acid filtrate of tissues

by ethanol and determined by an anthrone procedure previously used for
dextran and blood sugar.
3. Studies have shown that’ extracts made by boiling liver with 30 per
cent, KOH contain material that is not glycogcn. This material is dialyz-
able, anthrone-sensitive, and decomposed to some extent by prolonged
boiling in KOH solution, and, after hydrolysis, it, reduces alkaline copper
solution; it is, t’herefore, a source of error in glycogen methods based upon
alkali extraction of tissues.
4. The studies made indicated that the distinction between “free” and
“fixed” glycogen, reported in the literature, is an incorrect assumption.

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BIBLIOGRAPHY

1. Pfliiger, E. F. W., Das Glykogen und seine Beziehungen zur Zuckerkrankheit,

Bonn, 2nd edition (1905).
2. Good, C. A., Kramer, H., and Somogyi, M., J. Biol. C&m., 103, 485 (1933).
3. Cori, G. T., J. Biol. Chem., 96, 259 (1932).
4. Morris, D. I,., Science, 107, 254 (1948).
5. Seifter, S., Dayton, S., Novic, B., and Muntwyler, E., Arch. Biochem., 25, 191
(1950).
6. Bloom, W. L., Lewis, G. T., Schumpert, M. Z., and Shen, T.-M., J. BioZ. Chem.,
188, 631 (1951).
7. Hanson, It. W., Schwartz, H. S., andBarker, S. B., Abstracts, American Chemical
Society, 128th meeting, Minneapolis, 73C (1955).
8. Carroll, N. V., Longley, R. W., and Roe, J. H., Abstracts, American Chemical
Society, 127th meeting, Cincinnati, 23C (1955).
9. Roe, J. H., J. Biol. Chem., 208, 889 (1954).
10. Roe, J. H., J. Biol. Chem., 212, 335 (1955).
11. Willstiitter, R., and Rohdewald, M., 2. physiol. Chem., 226, 103 (1934).
12. Kemp, A., and Kits van Heijningen, A. J. M., Biochem. J., 66, 646 (1954).
13. Kits van Heijningen, A. J. M., and Kemp, A., Biochem. J., 59, 487 (1955).
THE DETERMINATION OF GLYCOGEN
IN LIVER AND MUSCLE BY USE OF
ANTHRONE REAGENT
Nicholas V. Carroll, Robert W. Longley and
Joseph H. Roe
J. Biol. Chem. 1956, 220:583-593.

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